This project is directed toward identifying the actual products of chemical reaction between alkylating agents which are mutagens and/or carcinogens and informational macromolecules; then assessing the role of various modifications in producing biological effects. Specifically, we plan to study the products formed and their fate during replication, when the powerful carcinogen, ethylnitrosourea, acts on DNA in rats. We know from our recent work that N-nitroso ethylating agents primarily alkylate oxygens (in vitro and in cultured human and rat cells), including the phosphodiesters and base oxygens of C, U, T and G (and in the case of RNA, the 2'-O of ribose) and several of these are mutagenic events. The effects of oxygen substitution on the physical and biological properties of nucleic acids will be studied in several ways. After determining the stability of O-alkyl derivatives under a variety of conditions, we have developed appropriate methods for quantitating all the O-alkyl bases, 7-EtG, 3-EtA and ethyl-phosphotriesters in enzyme digests of 14C-ethyl-nitrosourea-treated DNA with a total of 1-3 ethyl groups per 10 to the 5th power DNA-P. At this level of alkylation, there is little cytotoxicity and animal growth is normal so that excision of each alkyl derivative can also be quantitatively determined. Our objective is to find whether there is a correlation between rates of excision or persistence of O-alkyl products and organ susceptibility to neoplastic transformation. We will also prepare homopolymers and heteropolymers of O-alkyl pyrimidines and purines in order to study their secondary structure, transcription and translation. The effect of other substitutions resulting from vinyl chloride metabolites will also be studied by examining the ability of modified polymers to function as templates or messengers.